Nonribosomal peptide synthetases (NRPSs) are large multidomain enzymes responsible for the biosynthesis of many pharmacologically important bioactive compounds of great structural diversity [Marahiel et al., Chem. Rev. (Washington, D.C.) 97: 2651-2673 (1997); Schwarzer et al., Nat. Prod. Rep. 20: 275-287 (2003); Cane et al., Science 282, 63-68 (1998)]. Prominent examples are the antibiotics penicillin, vancomycin, and actinomycin D, the immunosuppressant cyclosporine A, the siderophore enterobactin, and the antitumor drug bleomycin. NRPSs are organized into distinct modules, each of them responsible for the incorporation of one amino acid into the nascent peptide chain. A module can be further subdivided into catalytic domains, which are responsible for the coordinated recognition and activation [adenylation (A) domain] [Stachelhaus et al., Chem. Biol. 6: 493-505 (1999)], covalent binding and transfer [peptidyl carrier protein (PCP) domain] [Stachelhaus et al., Chem. Biol. 3: 913-921 (1996)], and incorporation [condensation (C) domain] of a certain substrate amino acid into the peptide chain [Stachelhaus et al., J. Biol. Chem. 273: 22773-22781 (1998)]. In addition to these so-called core domains, optional domains catalyze the modification of incorporated residues, i.e., by epimerization (E) or N-methylation (MT) domains [Walsh et al., Curr. Opin. Chem. Biol. 5: 525-534 (2001)]. Product release is normally effected by a thioesterase (Te) domain, catalyzing the formation of linear, cyclic, or branched cyclic products, representative for the class of NRPSs [Trauger et al., Nature 407: 215-218 (2000)].
Because of the modular organization of NRPSs and the colinearity between biosynthetic template and product, the NRP assembly line mechanism accommodates an enormous potential for biocombinatorial approaches. Crucial for such approaches is a profound knowledge about the substrate selectivity of catalytic domains and the determinants of selective communication between modules. In this context, little is known about the intermolecular communication between NRPSs within the same biosynthetic complex [Hahn et al., Proceeding of the National Academy of Sciences (USA) 101(44): 15585-15590 (2004)].
Ecteinascidin 743 (ET-743, trabectedin, Yondelis®) is a chemotherapeutic natural product isolated from the Caribbean mangrove tunicate Ecteinascidia turbinata.(1) The compound has been designated an orphan drug in the United States and Europe for the treatment of soft tissue sarcoma and ovarian cancer, and it is currently undergoing phase II clinical trials for breast and pediatric cancers and phase III trials for soft tissue sarcoma. Previous work suggests that the drug is actually produced by the uncultivable bacterial symbiont Candidatus Endoecteinascidia frumentensis. Bacterial symbionts like E. frumentensis have long been proposed to be responsible for the production of biomedically intriguing natural products isolated from sessile invertebrate animals. However, the general inability to culture these organisms in the laboratory severely hinders the study of their biosynthetic pathways and the ability harness the full potential of their natural products. Although E. frumentensis remains incapable of being cultured in the laboratory, the chemotherapeutic potential of ET-743 has fueled over two decades of research since the compound's original discovery and isolation. The wealth of information provided in these studies in combination with the similarity of ET-743 to three well-characterized compounds derived from cultivable bacteria makes E. turbinata an ideal model system to develop a repertoire of tools to facilitate the study of uncultivable bacterial symbiont natural products.
Previous studies suggest that the ET-743 producer, E. frumentensis is actually an endosymbiont, living within tunicate host's cells. This hypothesis is supported by phylogenetic analysis depicting the intracellular pathogen Coxiella burnetii as a close relative of E. frumentensis. Furthermore, a previous study examining the location of the bacteria at different stages in the host's life cycle also suggested the producer to be an endosymbiont Moss et al., Mar Biol. 143: 99-110 (2003). Microscopic analysis of probe-stained cells and tissue revealed no lysing of the bacteria, no host pathology, nor endocytic markers, suggesting the bacteria's presence within the host cell was symbiotic in nature.
Obtaining sufficient amounts of ET-743 has presented a challenge since it was first isolated in 0.0001% yield from the natural source [Rinehart et al., The Journal of Organic Chemistry 55: 4512-4515 (1990)]. Aquaculture has proven to be viable [Fusetani N (ed): Drugs from the Sea. Basel. Karger. 2000. pp120-133. Chapter: Dominick Mendola Aquacultural Production of Bryostatin 1 and ecteinascidin 743; Fusetani, Drugs from the Sea. (2000); Carballo, Journal of the World Aquaculture Society 31: 481 (2000)], although not an economical method for supplying ET-743 for clinical trials and commercial use [Cuevas, Natural product reports 26: 322 (2009)]. Total synthesis of ET-743 was first reported [Corey et al., Journal of the American Chemical Society 118: 9202-9203 (1996)] and further routes of synthesis have been published [Endo et al., Journal of the American Chemical Society 124: 6552-6554 (2002), Chen et al., Journal of the American Chemical Society 128: 87-89 (2005), Zheng et al., Angewandte Chemie. International edition in English 45: 1754 (2006), and Fishlock et al., The Journal of Organic Chemistry 73: 9594-9600 (2008)]. Commercial production by PharmaMar has used a semi-synthetic scheme in which cyanosafracin B is transformed into ET-743 over eight steps [Cuevas et al., Organic Letters 2: 2545-2548 (2000)] as safracin B can be cultured on the kilogram scale from the wild-type producer Pseudomonas fluorescens [Ikeda, Journal of Antibiotics. Series B 36: 1290 (1983)]. The structural similarity of ET-743 to the tetrahydroisoquinoline alkaloid natural products saframycin C, saframycin MX1 and cyanosafracin (see Scheme 1, below), each derived from three distinct cultivable bacteria, indicated a prokaryotic origin for the tunicate-derived metabolite.
